Elisa Biondi

Laboratory evolution of artificially expanded DNA gives redesignable aptamers that target the toxic form of anthrax protective antigen

Reported here is a laboratory in vitro evolution (LIVE) experiment based on an artificially expanded genetic information system (AEGIS). This experiment delivers the first example of an AEGIS aptamer that binds to an isolated protein target, the first whose structural contact with its target has been outlined and the first to inhibit biologically important activities of its target, the protective antigen from Bacillus anthracis. We show how rational design based on secondary structure predictions can also direct the use of AEGIS to improve the stability and binding of the aptamer to its target. The final aptamer has a dissociation constant of ∼35 nM. These results illustrate the value of AEGIS-LIVE for those seeking to obtain receptors and ligands without the complexities of medicinal chemistry, and also challenge the biophysical community to develop new tools to analyze the spectroscopic signatures of new DNA folds that will emerge in synthetic genetic systems replacing standard DNA and RNA as platforms for LIVE.

Convergent donor and acceptor substrate utilization among kinase ribozymes

Accommodation of donor and acceptor substrates is critical to the catalysis of (thio)phosphoryl group transfer, but there has been no systematic study of donor nucleotide recognition by kinase ribozymes, and there is relatively little known about the structural requirements for phosphorylating internal 2′OH. To address these questions, new self-phosphorylating ribozymes were selected that utilize ATP(gammaS) or GTP(gammaS) for 2′OH (thio)phosphorylation. Eight independent sequence families were identified among 57 sequenced isolates. Kinetics, donor nucleotide recognition and secondary structures were analyzed for representatives from each family. Each ribozyme was highly specific for its cognate donor. Competition assays with nucleotide analogs showed a remarkable convergence of donor recognition requirements, with critical contributions to recognition provided by the Watson–Crick face of the nucleobase, lesser contributions from donor nucleotide ribose hydroxyls, and little or no contribution from the Hoogsteen face. Importantly, most ribozymes showed evidence of significant interaction with one or more donor phosphates, suggesting that—unlike most aptamers—these ribozymes use phosphate interactions to orient the gamma phosphate within the active site for in-line displacement. All but one of the mapped (thio)phosphorylation sites are on unpaired guanosines within internal bulges. Comparative structural analysis identified three loosely-defined consensus structural motifs for kinase ribozyme active sites.

A small ribozyme with dual-site kinase activity

Phosphoryl transfer onto backbone hydroxyls is a recognized catalytic activity of nucleic acids. We find that kinase ribozyme K28 possesses an unusually complex active site that promotes (thio)phosphorylation of two residues widely separated in primary sequence. After allowing the ribozyme to radiolabel itself by phosphoryl transfer from [γ-32P]GTP, DNAzyme-mediated cleavage yielded two radiolabeled cleavage fragments, indicating phosphorylation sites within each of the two cleavage fragments. These sites were mapped by alkaline digestion and primer extension pausing. Enzymatic digestion and mutational analysis identified nucleotides important for activity and established the active structure as being a constrained pseudoknot with unusual connectivity that may juxtapose the two reactive sites. Nuclease sensitivities for nucleotides near the pseudoknot core were altered in the presence of GTPγS, indicating donor-induced folding. The 5′ target site was more strongly favored in full-length ribozyme K28 (128 nt) than in truncated RNAs (58 nt). Electrophoretic mobilities of self-thiophosphorylated products on organomercurial gels are distinct from the 5′ mono-thiophosphorylated product produced by reaction with polynucleotide kinase, potentially indicating simultaneous labeling of both sites within individual RNA strands. Our evidence supports a single, compact structure with local dynamics, rather than global rearrangement, as being responsible for dual-site phosphorylation.

Lewis acid catalysis of phosphoryl transfer from a copper(II)-NTP complex in a kinase ribozyme

The chemical strategies used by ribozymes to enhance reaction rates are revealed in part from their metal ion and pH requirements. We find that kinase ribozyme K28(1-77)C, in contrast with previously characterized kinase ribozymes, requires Cu2+ for optimal catalysis of thiophosphoryl transfer from GTPγS. Phosphoryl transfer from GTP is greatly reduced in the absence of Cu2+, indicating a specific catalytic role independent of any potential interactions with the GTPγS thiophosphoryl group. In-line probing and ATPγS competition both argue against direct Cu2+ binding by RNA; rather, these data establish that Cu2+ enters the active site within a Cu2+•GTPγS or Cu2+•GTP chelation complex, and that Cu2+•nucleobase interactions further enforce Cu2+ selectivity and position the metal ion for Lewis acid catalysis. Replacing Mg2+ with [Co(NH3)6]3+ significantly reduced product yield, but not kobs, indicating that the role of inner-sphere Mg2+ coordination is structural rather than catalytic. Replacing Mg2+ with alkaline earths of increasing ionic radii (Ca2+, Sr2+ and Ba2+) gave lower yields and approximately linear rates of product accumulation. Finally, we observe that reaction rates increased with pH in log-linear fashion with an apparent pKa = 8.0 ± 0.1, indicating deprotonation in the rate-limiting step.

Separating and analyzing sulfur-containing RNAs with organomercury gels.

Polyacrylamide gel electrophoresis is a widely used technique for RNA analysis and purification. The polyacrylamide matrix is highly versatile for chemical derivitization, enabling facile exploitation of thio-mercury chemistry without the need of tedious manipulations and/or expensive coupling reagents, which often give low yields and side products. Here, we describe the use of [(N-acryloylamino)phenyl]mercuric chloride in three-layered polyacrylamide gels to detect, separate, quantify, and analyze sulfur-containing RNAs.

RNA Structural Analysis by Enzymatic Digestion

Enzymatic probing is a rapid, straightforward method for determining which regions of a folded RNA are structurally constrained. It can be carried out using very small amounts of material, and is especially suitable for short RNAs. Here we report a protocol that we have found to be useful and readily adaptable to the evaluation of RNAs up to 150-200 nucleotides in length. Considerations for optimization are also included. In brief, the method includes folding end-labeled RNA into its native conformation, partial digestion with structure-sensitive nucleases, and identification of the cleavage sites by electrophoretic separation of the cleavage fragments.

Mineral-Organic Interactions in Prebiotic Synthesis

A common criticism of “prebiotic chemistry research” is that it is done with starting materials that are too pure, in experiments that are too directed, to get results that are too scripted, under conditions that could never have existed on Earth. Planetary scientists in particular remark that these experiments often arise simply because a chemist has a “cool idea” and then pursues it without considering external factors, especially geological and planetary context. A growing literature addresses this criticism and is reviewed here. We assume a model where RNA emerged spontaneously from a prebiotic environment on early Earth, giving the planet its first access to Darwinism. This “RNA First Hypothesis” is not driven by the intrinsic prebiotic accessibility; quite the contrary, RNA is a “prebiotic chemist’s nightmare.” However, by assuming models for the accretion of the Earth, the formation of the Moon, and the acquisition of Earth’s “late veneer,” a reasonable geological model can be envisioned to deliver the organic precursors needed to form the nucleobases and ribose of RNA. A geological model having an environment with dry arid land under a carbon dioxide atmosphere receiving effluent from serpentinizing igneous rocks allows their conversion to nucleosides and nucleoside phosphates. Mineral elements including boron and molybdenum prevent organic material from devolving to form “tars” along the way. And dehydration and activation allows the formation of oligomeric RNA that can be stabilized by adsorption on available minerals.

Artificially Expanded Genetic Information Systems for New Aptamer Technologies

Directed evolution was first applied to diverse libraries of DNA and RNA molecules a quarter century ago in the hope of gaining technology that would allow the creation of receptors, ligands, and catalysts on demand. Despite isolated successes, the outputs of this technology have been somewhat disappointing, perhaps because the four building blocks of standard DNA and RNA have too little functionality to have versatile binding properties, and offer too little information density to fold unambiguously. This review covers the recent literature that seeks to create an improved platform to support laboratory Darwinism, one based on an artificially expanded genetic information system (AEGIS) that adds independently replicating nucleotide “letters” to the evolving “alphabet”.